Examining the influence of functionalized POSS on the structure and bioactivity of flexible polyurethane foams

Journal Pre-proof Examining the influence of functionalized POSS on the structure and bioactivity of flexible polyurethane foams Edyta Hebda, Artur Bukowczan, Sławomir Michałowski, Sebastian Wroński, Paulina Urbaniak, Mariusz Kaczmarek, Emilia Hutnik, Aleksandra Romaniuk, Maria WolunCholewa, Krzysztof Pielichowski PII:

S0928-4931(19)31792-8

DOI:

https://doi.org/10.1016/j.msec.2019.110370

Reference:

MSC 110370

To appear in:

Materials Science & Engineering C

Received Date: 20 May 2019 Revised Date:

25 October 2019

Accepted Date: 25 October 2019

Please cite this article as: E. Hebda, A. Bukowczan, Sł. Michałowski, S. Wroński, P. Urbaniak, M. Kaczmarek, E. Hutnik, A. Romaniuk, M. Wolun-Cholewa, K. Pielichowski, Examining the influence of functionalized POSS on the structure and bioactivity of flexible polyurethane foams, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110370. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Examining the influence of functionalized POSS on the structure and bioactivity of flexible polyurethane foams Edyta Hebdaa*, Artur Bukowczana, Sławomir Michałowskia, Sebastian Wrońskib, Paulina Urbaniakc, Mariusz Kaczmarekd, Emilia Hutnikd, Aleksandra Romaniuke, Maria Wolun-Cholewac, Krzysztof Pielichowskia

*

Corresponding author at: Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska Street, 31-155 Kraków, Poland E-mail address: [email protected] (E. Hebda)

a

Department of Chemistry and Technology of Polymers, Cracow University of Technology, 24 Warszawska Street, 31-155 Kraków, Poland

b

AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, al. 30 Mickiewicza Street, 30-059 Krakow, Poland c

Department of Cell Biology, Poznan University of Medical Sciences, 5D Rokietnicka Street, 60-806 Poznan, Poland

d

Department of Immunology, Chair of Pathomorphology and Clinical Immunology, Poznan University of Medical Sciences, 5D Rokietnicka Street, 60-806 Poznan, Poland e

Department of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, 49 Przybyszewskiego Street, 60-355 Poznan, Poland

ABSTRACT This work reports for the first time on a new class of flexible polyurethane foam hybrids (PUFs) synthesized with the use of less toxic aliphatic hexamethylene diisocyanate (HDI), which have been chemically modified by POSS moieties. The flexible polyurethane foam hybrids (PUFs) chemically modified by functionalized polyhedral oligomeric silsesquioxanes: octa(3-hydroxy-3methylbutyldimethylsiloxy)POSS (OCTA-POSS) and 1,2-propanediolizo-butylPOSS (PHIPOSS), was obtained. The resulting foams, which contain 0 to 15 wt. % POSS, were characterized in terms of their structure, morphology, density and compressive strength. The FTIR results indicate the chemical incorporation of both OCTAPOSS and PHIPOSS into the polyurethane matrix. SEM-EDS analysis showed that both OCTAPOSS and PHIPOSS nanoparticles are distributed homogeneously in the foam structure; at 15 wt. % load PHIPOSS characteristic "crosses" are formed. With the increase of PHIPOSS content in the matrix, the formation of agglomerates is observed, as revealed by WAXD spectra. The introduction of POSS compounds reduces the porosity of the polyurethane, with the number of pores increasing as the

amount of modifier increases. Mechanical tests - compressive strength - show that the hardness of modified materials (5 wt. % POSS) increases compared to the reference material. An incubation was carried out in a simulated physiological fluid (SBF) to pre-assess the bioactivity of the materials obtained. The obtained results confirmed the formation of a hydroxyapatite layer on the PUF-POSS surface. Cytotoxicity, cell cycle and apoptosis of osteoblast cells and fibroblasts were assessed in the presence of the PUF-POSS materials. Test materials have a cytotoxic effect on both established cell lines. PUF-PHIPOSS samples showed better biocompatibility than reference and PUF-OCTAPOSS samples, as they caused lower mortality of the examined cells. Keywords: flexible polyurethane foams, polyhedral oligosilsesquioxane, POSS, hybrid materials, bioactivity, cytotoxicity. 1. Introduction Polyurethanes (PU) are an important and dynamically growing group of polymeric materials that are manufactured in the form of foams, elastomers, coatings, adhesives and fibers. A special position among PU products is occupied by rigid, semi-rigid and flexible polyurethane foams. Their low mass and apparent density, while maintaining excellent mechanical and thermal insulating properties, make them attractive materials in a number of applications, including those in the biomedical field as drug release systems, scaffolds, stents, etc. PU are prepared by the stepwise polyaddition reaction of isocyanates and compounds containing hydroxyl groups, in which the reaction mixture is foamed using blowing agents. Flexible polyurethane foams have open cells, with walls separating adjacent cells ruptured during the foam formation process. [1] The foaming process is divided into four stages: (I) mixing of the monomers and bubble nucleation; (II) rise of foam; (III) phase separation and cell opening; and (IV) formation of a foam. The polymer matrix of flexible PU foams has a segmented structure with separated flexible polyether phase and hard urea domains [2-4]. In the case of flexible polyurethane foams, the gas entrapped in the closed cells also resists compression, reducing the cushioning quality of the foam. Thus, it is crucial to have nearly 100% open cells in flexible polyurethane foams. Control of the cell-opening process is thus important in the production of PU foams [5]. Flexible PU foams are block copolymers that owe their elastic properties to the phase separation of so-called “hard blocks” and “soft blocks”. Hard blocks are rigid structures that are physically cross-linked and give the polymer its firmness while soft blocks are stretchable chains that give the polymer its elasticity. Therefore, polyurethane foams can be customized by adapting the composition and the ratio of these blocks [6, 7]. Another way to improve flexible PU foams properties, especially mechanical and acoustic properties, is by incorporating fillers, such as dolomite (CaMg(CO3)2), calcium carbonate [8], fumed silica [9], nanoclay or MWNTs [10] are added to PU-flexible foams for improving mechanical properties. An interesting group of hybrid organic-inorganic materials are polyhedral oligomeric silsesquioxanes, POSS. Silsesquioxanes are a class of hybrid organosilicon materials - with the general formula RSiO3/2 [11] - containing a three-dimensional, chemically substituted and thermally stable silicon-oxygen core. POSS structure is differentiated not only due to the variable number of silicon atoms included in the silicon-oxygen cage, but also the number and type of substituents attached to it. These substituents are basically divided into two groups: reactive and non-reactive. It is important that as a result of the appropriate selection of functional groups connected with peripheral silicon atoms in the cage of silsesquioxane, which constitute approx. 70% of the total volume of the POSS molecule, one can design and control the properties of the obtained material [12, 13].

The main advantages of silsesquioxanes are non-toxicity, high mechanical strength, solubility in most organic solvents, thermal stability and considerable resistance to atmospheric conditions [14-16]. The unique POSS cage structure, ease of modeling and introduction them into polymer matrices favour the use of silsesquioxanes within the application biomedical. The main features which determine the medical destiny of POSS are biocompatibility, biodegradability, cytological compatibility, and lack of toxicity and thermodynamic stability. Thanks to these properties, POSS nanoparticles can be used for encapsulation of drugs, as well as in dental and tissue engineering [17-19]. Poly(carbonate–urea)urethane (PCU) reinforced with POSS were tested as materials for artificial capillaries [20, 21] and for artificial heart valve [22] It has been found that the introduction of POSS nanoparticles as side groups of the poly(carbonate-urethane) macro chain causes a significant improvement in mechanical properties (tensile and tear strength) of PCU-POSS nanocomposites. In the course of other work, Ghanbari et al. [23] showed that the presence of POSS compounds reduces the tendency of PCU to calcification and thus extends the duration of use of these materials in vivo. Wang and co-workers introduced silsesquioxane into polyurethane elastomers to reduce surface tension [24, 25] and to reduce bacterial adhesion [26]. In our previous studies [27-29], we investigated the influence of POSS nanoparticles on macromolecular architectures of rigid polyurethane foams. This work reports for the first time, the effect of the addition of POSS particles on the structure and properties of flexible aliphatic hexamethylene diisocyanate (HDI)-based polyurethane foams. Noteworthy, HDI shows less biological toxicity compared to commonly used aromatic isocyanates, which is of primary importance for biomedical products. 2. Materials and method 2.1 Reagents Flexible polyurethane foams were obtained from a mixture of polyetherols under the commercial names Rokopol F3600 (hydroxyl number 47 mg KOH/g, the viscosity at 25oC is 580 mPas, and the average molecular weight 3600 g/mol, PCC Rokita SA, Poland) and RF551 (number hydroxyl 440 mg KOH/g, the viscosity at 25oC is 3000-5000 mPas, and the average molecular weight 600 g/mol, PCC Rokita SA, Poland). Polyol F3600 is an ethoxylated and propoxylated glycerol-based triol, while polyol RF551 is polyoxyalkylene multi-hydroxyl alcohol. The isocyanate component was hexamethylene diisocyanate (HDI) with 49% of NCO groups (SigmaAldrich). The catalyst in the process was Dabco T9 catalytic system (stannous octoate) and Dabco BL11 (N, N, N ', N'-tetramethyl-2,2'oxybis (ethylamine) and oxydipropanol) supplied by Air Products. A silicone surfactant L627 was used as the foam structure stabilizer (Momentive Performancce Materials Inc., Czech Republic). The blowing agent was water. As the polyurethane foam modifiers, 1,2-propanediolisobutylPOSS (PHIPOSS) and octa (3hydroxy-3-methylbutyldimethylsiloxy) POSS (OCTAPOSS) were used which were provided by Hybrid Plastics (USA). 2.2 Preparation of PU foams Flexible polyurethane foams were produced on a laboratory scale using the one-stage method from a two-component system, with an equivalent ratio of NCO to OH groups equal to 1:1. Component A was obtained as a result of thorough mixing of Rokopol F3000 and RF551 in a mass ratio of 1:1, surfactant, catalysts, blowing agent and - in the case of modified foams - an appropriate amount of modifier (5, 10 or 15 wt% POSS vs polyol). Component B was hexamethylene diisocyanate. Components A and B were mixed and poured into a Teflon open

mold and foam was expanded. Flexible polyurethane foams after having been left to the end of the growth process for 1 hour, and then conditioned at room temperature for at least 24 hours. The detailed procedure was described in [29]. 2.3 Characterization 2.3.1 Chemical, morphological, physical and mechanical characterizations The apparent density of foams was determined in accordance with PN-EN ISO 845. The mass of the samples was determined by means of an electronic analytical balance with an accuracy of 0.1 mg, and the volume - as a result of dimensioning of rectangular samples using a caliper, with an accuracy of 0.1 mm. The chemical structure of the obtained PUR foams was confirmed by infrared spectroscopy (FT-IR), using a Nicolet iS5 spectrometer equipped with a diamond crystal attenuated total reflectance unit by Thermo Electron Corporation. For each material, the measurement was performed 5 times in different places. The obtained results were averaged using the Omnic computer program. Wide angle X ray diffraction (WAXD) investigations were performed by applying a Bruker D Phaser diffractometer in the reflection mode. A standard Cu Kα anode with wavelength λ = 1.54184 Å was used. The microstructure of the foams was characterized by scanning electron microscopy (SEM), using a JEOL InTouch Scope JSM-6010LV microscope with energy-dispersive X-ray analysis capabilities, operated at 10 kV accelerating voltage. The micro tomography measurements presented in this paper were performed using “Nanotom 180N" device produced by GE Sensing & Inspection Technologies Phoenix X-ray. The machine is equipped with nanofocus X-ray tube with maximum 180kV voltage. The tomograms were registered on Hamamatsu 2300x2300 pixel detector. Measured objects were reconstructed with the aid of proprietary GE software datosX ver. 2.1.0 with use of Feldkamp algorithm for cone beam X-ray CT [30]. All examined specimens were scanned at 60 kV of source voltage and 310 µA, with a rotation of the specimen of 360 degrees in 1600 steps. The exposure time was 500ms and a frame averaging of 5 and image skip of 1 was applied, resulting in a scanning time of 80 minutes. The reconstructed images had a voxel size of (4,0 µm)³.The post reconstruction data treatment was performed using free Fiji software [31] with BoneJ plugin [32]. Compressive strength was determined using a Zwick/Roell Z005 testing machine, in accordance with ISO 844, up to 40% deformation of the sample. The deformation speed was 10 mm/min. 2.3.2 Bioactivity test PUF samples were immersed in SBF solution, which contain concentrations of Ca2+ and PO43ions nearly equal to human blood plasma. The SBF solution was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4ˑ3H2O, MgCl2ˑ6H2O, CaCl2 and Na2SO4 (Sigma-Aldrich) in distilled water. This solution was buffered with tris (hydroxymethyl) aminomethane and adjusting pH at 7.45 at 37 oC by stirring and titrating it with HCl. 2.3.3 In vitro cytocompatibility tests Cell culture conditions

In our study we used an adherent established cell line of human osteoblast (osteosarcoma) U-2, derived from ATCC (ATCC®HTB-96 ™). Cells were cultured in RPMI-1640 media (Lonza), supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% Antibiotic Antimycotic Solution (10,000 U penicillin, 10 mg/ml streptomycin, 25 mg/ml amphotericin B; SIGMAAldrich, Merck, St. Louis, MO, USA). Cells were cultured on plastic plates in an incubator, under standard conditions (37°C, 5% CO2 atmosphere and 95% humidity) until they reached 90% of confluence. Then cells were washed with phosphate buffer (PBS), harvested from plates with a trypsin enzyme (0.25% Trypsin-EDTA; Sigma) solution and counted using a hemocytometer (Fuchs-Rosenthal’s hematologic camera). Finally, cells prepared in this way, were applied to the surfaces of the test materials. Test samples preparation Test samples were placed in 24-well culture plates, filled with 0,5ml of culture medium and preincubated in cell culture conditions for 24 hours. The VOLUME area of test samples was approximately 1cm3. Afterwards cell culture medium was removed, 105 of osteoblasts cells per well, suspended in 1ml of culture medium, were applied directly on each test material surface. The control cells were cultured under the same conditions without any contact with tested samples. Cells were cultured for 24, 48 and 72 hours. All analyses were performed in a chamber with laminar air flow and disposable equipment were used to ensure sterile conditions. Microscopic analysis In order to perform morphological analysis of cells, confocal microscope was used to obtain photos in high resolution. After 24 hours of growth on the tested samples surface, cells were imaged with LSM 780 Zeiss confocal microscope. Microscope analysis were performed followed by osteoblasts nucleus staining with Hoechst/Propidium Iodide. Blue-fluorescent Hoechst dye penetrates the cellular membrane and stains the chromatin of nonapoptotic cells, and redfluorescent Propidium Iodide dye stains (via intercalation into DNA) dead cells nuclei in pink/red. MTT test (cell viability assay) The cytotoxicity level was assessed by determining the percentage of dead cells as well as their growth inhibition degree. In order to evaluate the cytotoxicity of the materials tested the MTT assay was performed. The MTT assay is a quantitative and sensitive detection of cell proliferation as it measures the growth rate of cells by virtue of a linear relationship between cell activity and absorbance. MTT cell-proliferation assay measures the reduction of water-soluble MTT tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a blue– purple insoluble formazan crystals, by viable cells mitochondria (as it requires presence of the active mitochondrial dehydrogenase). The formazan must be solubilized prior to recording absorbance readings, thus formazan crystals are extracted from the cells with a solubilizing solution (10 % SDS in 0.01 M HCl). At the time of analyses U-2 OS osteoblasts were cultured directly on material samples located in the complete culture media (supplemented as described above), in 24-well culture plate, for 6, 12 and 24 hours. Control group consisted of cells without any contact with tested samples. After specified time periods 10µl of MTT solution (5 mg/ml thiazolyl blue Tetrazolium Bromide) were applied. Followed by 4 hours incubation (standard conditions), the formazan crystals were released from the cells with 100 µl of a solubilizing solution. The absorbance measurements were carried out spectrophotometrically, on microplate reader (Multiscan, Labsystems, Thermo Fisher Scientific Inc., U.K), at 570nm and 690nm wavelengths. Test results were given as RVC (Relative Viability of Cells), which is defined as correlation of absorbance value for cultures with direct or indirect contact with the materials tested, to

absorbance value of cells growing directly on culture plate surface without any material (control samples). Finally, the viability of cells growing on the tested material was given in relation to the viability of cells growing directly on culture plate surface without any material (control samples). Results were presented as a Relative Viability of Cells value. The RVC value was calculated from the formula: RVC(%)=[(a-b)/(c-b)]x100, where: a- absorbance of the tested sample; b-absorbance of the blank control (reaction without the cells); c- absorbance of the control grown without any material. Cell cycle evaluation Cells were detached from the samples surface after 24, 48 and 72 hours of culturing, with trypsin solution. All of the tested samples were transferred to a new 24-well plate and suspended in 0,5ml of trypsin solution, in order to detach the cells growing directly on their surface. Simultaneously, cells growing on the plate surface, which were not directly attached to the samples, were harvested with 0,5ml of trypsin. All of the cells were incubated for 5 minutes. PBS buffer was used in order to deactivate trypsin enzyme. Cells were washed twice with PBS and centrifuged for 5 minutes at 1500rpm. Subsequently, cells were suspended in Permeabilization Buffer (Perm/Wash; Becton Dickinson) and incubated for 30 minutes at 4°C. After additional washing step with PBS, the cell pellets were stained with a 1mg/ml propidium iodide solution (PI, Sigma). Incubation with PI and was performed for 30minutes at 37°C in darkness. Stained cells were assessed by FACS Canto flow cytofluorimeter (BD Biosciences). Analysis results presented as histograms, were evaluated by FACS Diva software (Becton Dickinson), integrated with the flow cytometer. Mean fluorescent intensity (MFI) emitted by PI is directly proportional to the amount of nuclear DNA, depending on the phase of the cell cycle. The percentage of cells in the S phase of the cell cycle indicate the number of replicating cells which is equal to proliferative activity of tested cells. Moreover, cytometric histograms allow for determination of the percentage of cells in the G2/M phase (preceding the mitosis) and percentage of dead cells. Apoptosis evaluation with Annexin V Apoptosis evaluation was performed using a commercially available FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). The procedure was performed according to the protocol attached. In the first step cells were resuspended in 100µl of Annexin buffer (1x). Afterwards 5µl of propidium iodide and 5µl of Annexin V conjugated with fluorescein were added, tittered and incubated for 15 minutes in dark. Eventually 160µl of Annexin buffer (1x) was added to each test tube. All of the analysis were performed analogously to the cell cycle analysis. Cells were defined as necrotic, in early or late apoptosis, depending on the of FITC fluorescence and/or propidium iodide (PI) proportion. 3. Results and discussion 3.1 Structure and morphology of composite foams Fig. 1 shows the spectra of pure OCTAPOSS and PHIPOSS, the reference flexible foam (PUF), and the hybrid foams containing 15 wt% of POSS. The spectra were used to characterize the chemical structures of neat and modified polyurethane foams (PUF).

Fig. 1. ATR-FTIR spectra with the PUF-OCTAPOSS [A] and PUF-PHIPOSS [B]. Structural investigations of the PU POSS hybrid composites by IR spectroscopy revealed the characteristic absorptions bands at 3334 cm-1 corresponding to the stretching vibrations of N-H. The peak at about 2934 cm-1 and 2863 cm-1 correspond to the symmetric and asymmetric stretching vibration of C-H in hydrocarbon chains, respectively. Peak with a maximum of 1694 cm-1 is derived from stretching vibrations associated with the occurrence of the C=O group. Also visible are bands originating from bending vibrations of N-H (1560 cm-1) bonds and asymmetric stretching vibrations of C-O-C (1094 cm-1) groups [33, 34]. No absorption band was observed at 2240 cm-1 assigned to NCO groups in the spectra of the PU foams, which indicated that the isocyanate group was completely consumed with the production of urethane or urea linkages. In the OCTAPOSS spectrum we observed the maximum absorbance corresponding to the wavenumber 1190 cm-1 derived from stretching of C-O in tertiary alcohols functional groups of OCTAPOSS. The lack of this band on the PUF-OCTAPOSS spectrum indicates the reaction of OCTAPOSS in the polyurethane foam. On the PUF-OCTAPOSS spectra there is a weak peak at the 885 cm-1 wavenumber associated with stretching vibrations of Si-O bonds. Another peak with a maximum of 853 cm-1 corresponds to the vibration of Si-CH3 in OCTAPOSS. With a wavenumbers of 773 and 779 cm-1, a peaks are visible that comes from the stretching vibrations of Si-C (Si-CH2 or Si-CH3) bonds occurring in the side groups in both OCTAPOSS and PHIPOSS respectively. In PUF-PHIPOSS hybrid no peak was observed at 691 cm-1. This band corresponds to a bending vibration of COH group in PHIPOSS. This band disappears in the spectrum of PUF matrix modified of PHIPOSS (15 wt%). At 470 cm-1, a peak is assigned to the bending vibrations from the COC present in the side group PHIPOSS. The obtained IR results confirm that POSS moieties were chemically built into the polyurethane structure to form PU-POSS hybrid materials. The cellular structure of the produced flexible polyurethane foams was evaluated on the basis of the microphotographs made by SEM technique. It was observed that the analyzed materials have an open cell structure, the cells closely adhere to each other and their shape is spherical. The addition of the filler to the polyurethane matrix results in a change in the size of the pores present in the sample. SEM morphology of the PUF-OCTAPOSS (Fig. 2 [B-D]) shows that the presence of a nanofiller in the system causes an increase in the size of cells in the material. Mapping of OCTAPOSS modified materials showed an even distribution of silicon on the surface of the tested material. There are few, blurred areas of OCTAPOSS agglomerates, however, they are not noticeable on WAXD patterns. The OCTAPOSS agglomerates are randomly distributed and their

size depends on the amount of nanofiller added. The greater the proportion of the nanofiller in the material, the larger the clusters of agglomerates. In Fig. 2 [E-G] SEM micrographs of PUR-PHIPOSS monohybrids are showed. These materials also show an open cell structure with spherical cells. In the SEM images for PUF-PHIPOSS, it can be seen that more and more smaller cells are formed. In Figure 2I, as in the case of PUFOCTAPOSS, in the PUF-PHIPOSS too, silicon is regulary distributed throughout the sample volume. However, in this case, numerous crystals in the shape of characteristic "crosses" are formed. The arm of such a crystal has a size of approx. 10 - 15 µm. Mapping of PUF-PHIPOSS foam showed the formation of a larger number of "crosses" with the increase of the content of the additive. It can be observed that agglomerates of PHIPOSS crystals are distributed throughout the entire volume of the material.

Fig. 2. SEM images of polyurethane foams [A-H: all images have the same magnification. x80, 200um, I: magnification x170, 100um, x1500, 10um and EDS mapping where show SEI images linked with silicon map].

To better characterize the microstructure of cellular material, X-ray computer micro-tomography (µ-CT) - a non-invasive three-dimensional (3D) imaging technique, was used. Three-dimensional visualization of reference foam for different additive concentrations is presented on Fig. 3.

Fig. 3. Three-dimensional visualization of reference foam [A], PUF-OCTAPOSS 15% [B] and PUFPHIPOSS 15% [C] concentration of the additive.

Based on tomographic data, the porosity and number of cells (in examined volume 7.2x7.2x8mm) versus concentration of the additive was determined. The result are presented in Fig. 4. Analysis of the results showed that the addition of the OCTA content has a significant influence on porosity of the obtained material. The highest porosity is observed for the 5% concentration, and decreases slightly with increasing concentration of additive. Also with the PHI admixture, the filler effect is observed, however, it is not significant. Only a small decrease in porosity was observed. The calculation of pore number in the sample volume, showed that with the increase of additive volume in sample, the number of pores initially decreases in comparison to the reference sample (without additive). Only after exceeding 10%, the number of pores increases again to a slightly higher value than for the reference sample. The charts of numbers of pores vs. additive concentration have similar trends for both materials (doped by PHI and OCTA).

Fig. 4. Porosity and number of cells versus OCTAPOSS and PHIPOSS concentration in polyurethane foams.

To examine the structure changes as a result of doping, two indices: mean thickness of analyzed structure, i.e. trabecular thickness (Tb. Th) and thickness of the background, i.e. trabecular spacing (Tb. Sp), were analyzed [35]. For Tb.Th the plug-in determines a diameter of the greatest sphere that fits within the analyzed structure. Similarly, when estimating Tb. Sp the voxels of background are filled with maximal spheres. The resulting thickness map, which represents the

3D distribution of computed pore diameters for different concentration of the additive is presented in Fig. 5.

Fig. 5. Map representing the 3D distribution of computed pore diameters for reference foam and for foam with different concentration of the additive.

The results obtained confirm the influence of additives on the porosity and the number of pores. It is clearly visible that the additives leads to the several changes in structure of the material, which becomes inhomogeneous, what is particularly visible for the sample with OCTA component. Large pores appear within the material structure. Increase of OCTA concentration up to 15%, leads to a heterogeneity decrease, which is associated with an increasing pore numbers in examined volume. A similar effect is observed in the sample with a PHI additive, however, in this case the changes in the structure are not significant. In the following step, for each of the analyzed samples, 3D data were used to derive pore size distribution. Corresponding histograms are presented in Fig. 6. These histograms confirm the previously observed trends of changes. For a PHI doped sample, the histograms were changed only slightly. The histograms for the reference and 5 and 10% doping samples are very similar. Only for the 15% PHI sample, the histogram shift towards the lower values is observed.

Fig. 6. Distribution of pores diameter for all examined samples.

The mean pore size values for the reference sample were 0.476 mm and for the PHI doped samples 5%, 10% and 15% - 0.549 mm, 0.494 mm and 0.475 mm respectively. In case of OCTA doped sample, for 5 and 10% of concentration, the histograms were significantly blurred and shifted towards higher values relatively to the reference sample. Once the additive content exceeds 15 wt%, the shape of the histograms changes, in particular the histograms become skewed left (there is a shift toward lower values). Average pore size values for 5, 10 and 15% OCTA doped samples were 0.699 mm, 0.705 mm and 0.671 mm respectively. The WAXD diffractograms of pure PHIPOSS and OCTAPOSS silsesquioxanes as well as unmodified PUF polyurethane, shown in Fig. 7, reveal the difference in the structure of PUFPHIPOSS and PUF-OCTAPOSS system components. Pure silsesquioxanes PHIPOSS and OCTAPOSS are high-crystalline substances, as indicated by the numerous and very sharp diffraction peaks present on the WAXD diffraction pattern. On the WAXD diffractogram of pure PUF is only wide of diffuse scattering maxima, which indicates the existence, at the molecular level of this polymer, of ordering only the short range, and thus the amorphous internal structure of both the soft and hard polyurethane phase.

Fig. 7. WAXD patterns recorded with the pure PUF, the hybrids and the POSS nanoparticles: [A] OCTA and [B] PHIPOSS and an exemplary deconvolution of WAXD pattern of pure PUF.

Due to the two-phase the supramolecular structure of PUF, it can be supposed that the distinct asymmetry of the main halo of amorphous polyurethane in the range of angles 2θ = 12-30 is due to the fact that it is composed of two maxima. Figure 7 shows the deconvolution of the WAXD patterns on the diffraction peak components of the reference polyurethane. One maximum corresponds to the distance between fragments of rigid segments in the hard phase (Fig. 7, diffraction maximum C), while the second distances correspond between fragments of flexible segments in the soft polyurethane phase (Fig. 7, maximum diffraction B). The presented distributions show that beside to the main amorphous halo there are two diffraction maxima with a much lower intensity located at an angle of 2θ equal to: 1) 8.8-12.2o - this is the prepeak of the main amorphous halo associated with the dispersion of some rigid segments in the soft phase domains (Fig. 7, diffraction maximum A), 2) 37.1-41.4o - its presence is related to the distances of atomic groups in the polyurethane chain (Fig. 7, diffraction maximum D) [36]. The incorporation of OCTAPOSS molecules into the polyurethane structure as nodes of the polymer network, regardless of the content introduced, did not result in the appearance of additional diffraction peaks on the obtained WAXD diffractograms (Fig. 7a). The amorphous nature of PUF-OCTAPOSS shows that OCTAPOSS is evenly dispersed in the polymer and reacted without forming crystals [37], however, the SEM-EDS analysis shows that clusters of OCTAPOSS agglomerates appear in the PUF-OCTAPOSS sample. This discrepancy may be due to too low sensitivity of the X-ray apparatus to detect OCTAPOSS agglomerates in the foam material. It is also observed a significant flattening and shifting of the main amorphous halo towards in the higher angles direction at the content of 15% OCTAPOSS; from 2θ = 19.4 for the reference polyurethane to 2θ = 20.5 for PUF-OCTAPOSS (15 wt%) as a result of the increase in interfacial distances through the greater packing of OCTAPOSS in PUF. Different results were recorded when PHIPOSS molecules are built into the polyurethane structure. When the PHIPOSS content in the PUF is 10% or 15wt%, on the diffractogram, reflexes appear at the deflection angle of 2θ = 8.1 ° (Fig. 7b), corresponding to the most intense diffraction peak of PHIPOSS, which confirms the presence of its crystallites. This showed the very high ability of PHIPOSS molecules to self-organize into a separate crystalline phase in the obtained PUF-PHIPOSS materials [36], which is confirmed by micrographs and silicon mapping in the SEM-EDS investigation. 3.2 Apparent density and compressive strength Apparent density is an important parameter describing the physical properties of the material and is one of the most important features determining the mechanical properties of foams (Fig. 8) Density of polymer foam depends on the amount of the material making up foam network, the density of the material making up the matrix of the foam and the density of the gas in cells [38, 39].

Fig. 8. Apparent density and compressive strength of the reference and foams modified by OCTA-POSS or PHI-POSS.

The density of the pure PUF was 51.6 kg/m3. For 5 wt% of PHIPOSS and OCTAPOSS hybrids, there was slight an increase in the foam density to 53,6 and 52.8 kg/m3, respectively. In the case of 10% of the OCTAPOSS additive, the density remains at a comparable level as with the content of 5wt% OCTAPOSS. A significant change is observable with 10 wt% of PHIPOSS in polyurethane foam and is 65.7 kg/m3. The highest density increase occurred at 15 wt% OCTAPOSS and PHIPOSS and amounted to 66.8 and 65.2 kg/m3, respectively. The increase in the density of foams containing a nanofiller can be associated with an increase in the viscosity of the polyol in which POSS is dispersed. The addition of POSS to the polyol causes the viscosity to increase, as a result of which the foaming process may be hindered, which will result in the formation of foams with a higher apparent density. The increase in the analyzed parameter may also result from the POSS tendency to crystallize in the polyurethane matrix, as shown by the WAXD spectra. The obtained materials were subjected to a compressive strength test, where the foams were submitted to the compression force deflection necessary to cause a deformation of 40% of the original size. This parameter quantifies the compression that a flexible foam supports without a significant loss in its morphology. The higher the compression force deflection 40 value, the more difficult the compression becomes because the foam is harder. Fig. 8 shows the graph obtained of compression force deflection 40 as a function of filler concentration for foams with POSS. The research shows that the hardness of OCTAPOSS and PHIPOSS modified materials (5wt%) increases on average by 0.46 and 0.21 kPa in relation to pure PUF hardness, respectively. The introduction of POSS in an amount of 10 and 15 wt% to the polyurethane matrix contributes to a decrease in hardness and only for OCTAPOSS this parameter is still higher compared to pure PUF. In the case of PUF-PHIPOSS (15 wt%), the average hardness drops by 0.31 kPa compared to pure PUF. The initial increase in hardness may result from the way POSS nanoparticles attach to the polymer matrix. The more branched the polymer chain is, the harder the material is. This dependence can be observed in the case of OCTAPOSS, which is incorporated into the polyurethane matrix as a network node, because it has as many as 8 functional groups. The addition of more OCTAPOSS (10 and 15 wt%) to the matrix results in a lower hardness due to the formation of POSS agglomerates. PHIPOSS, on the other hand, has two functional groups and joins the main chain as a side branch. The WAXD and

SEM-EDS studies show that in the case of the PHIPOSS addition, the degree of crystallinity of the tested materials increases. The increase in the size of the crystals causes the smaller cells to crack in favor of the formation of larger cells, and thus the hardness of the obtained PUFPHIPOSS materials. 3.3 Bioactivity and in vitro cytocompatibility of composite foams Bioactivity is defined by literature as the ability of material to elicit a specific response at the tissue-implant interface resulting in chemical bond formation between material and the living tissue [40]. The obtained foams were incubated in freshly prepared SBF (Simulated Body Fluid) solution with similar concentrations of inorganic salt ions to those which they are found in the plasma of human blood in accordance with the recipe described in the article [41]. The formation of apatites on the surface of the material as a result of incubation in SBF is an initial assessment of bioactivity before in vivo investigations. The samples were incubated for 4 weeks at a constant temperature of 37 °C. The SBF solution was replaced every 5 days, after which the surface was observed using a scanning electron microscope – Fig. 9. On the surface of all samples tested except the pure polyurethane, apatites were found after incubation in SBF.

Fig. 9. SEM images of PUF-OCTAPOSS(15%) [A] and PUF-PHIPOSS(15%) [B] foams mineralized in SBF for 4 weeks.

Numerous spherical hydroxyapatite (HA) granules have been observed, which the number and size increase with the increase in the content of POSS molecules in foams. There are empty places between the globules in which the HA does not precipitate. Together with the prolonged incubation time of samples in SBF fluid, globules begin to form a compact hydroxyapatite layer of varying thickness. Foams containing OCTAPOSS had tended to accelerate and evenly increase apatite, whereas in foams with PHIPOSS, this increase was slower. Nucleation and adhesion of hydroxyapatite on the surface of PU-POSS nanocomposites under in vitro conditions depends on the molar ratio Ca/P of the layers formed. SEM-EDS analysis of the materials after incubation in SBF was performed and the Ca/P ratio was determined. For all materials doped with POSS the Ca/P ratio was in the range of 1.54-1.63. In these investigations, the positive effect of POSS on the bioactivity of the obtained foam composites was confirmed.

In the next stage of the research, the obtained materials were examined to evaluate their cytotoxicity, relative to osteoblast and fibroblasts cells. Confocal microscopy Confocal microscope observations showed highly increased apoptosis of cells grown in contact with samples of materials tested. Most of the cells observed, were stained in red, due to the fluorescence of propidium iodide absorbed. What is more there were only single cells observed in the field of view – Fig. 10. Those observations confirmed the highly increased apoptosis ratio observed in cell cycle analysis.

Fig. 10. U-2 OS osteoblast cells growing on the surface of the tested samples. Cell nuclei stained in red with propidium iodide.

MTT test (cell viability assay) Cytotoxicity of tested materials was determined after 6, 12 and 24 hours of U-2 OS osteoblasts culturing. Control cells were grown without any contact with tested materials, directly on the culture plate. U-2 OS osteoblasts viability after 6 hours of culturing with samples of all tested materials was higher than in control. After 12 hours of culture, all samples showed a significant decrease in cell viability, which was lower than in the control. Cell survival ratio in case of cells cultured on the surface of all materials tested, after 24 hours was reduced to zero. For cells grown in contact with a sample of the reference material, the highest viability of osteoblasts was observed after 6 hour of culture in contact with samples of 15% OCT and 15% PHI. This lifetime decreased after 12 hours of culture and reached a value close to the viability of cells cultured in contact with the reference sample.

Cell cycle assessment Cell cycle analysis were subjected to U-2 OS osteoblast cells after 24, 48 and 72 hours of culturing for both-cells growing on tested materials surface and cells growing in their surroundings. The percentage of cells in the individual phases of the cycle, G0/G1, S and G2/M, and apoptotic cells were determined. Cell cycle analysis, showed higher level of osteoblast mortality in case of cells grown on the tested samples than in the case of culture in the surrounding of the materials. Control cells cultured without the material samples showed regular and balanced growth. After 48 hours of culture, the mortality of osteoblasts increased significantly. After 72 hours, there were only few cells displaying viability characteristics. All of the materials tested had a negative impact on the U-2 OS osteoblasts cell cycles. However the additives to the samples seem to reduce this effect, thus these samples impact was less aggressive. The highest percentage of dead cells was found within cells grown on the samples surface after 48 hours of culture in direct contact with the test samples. Cell death occurred less frequently in case of cells growing in the surrounding of the materials tested, however, along with the prolonged culture time, the number of dead cells gradually increased. The highest death cells index was observed for cells cultured in direct contact with OCTAPOSS modified samples. The smallest percentage of dead cells was observed in contact with a 5% PHIPOSS sample after 24 hours. After 48 hours, the predominant number of cells grown in direct contact with the reference sample, as well as the 5% and 10% OCTAPOSS samples were dead. Likewise in case of direct contact with the materials tested, after 48 hours of culture all of the tested samples decreased the osteoblasts growth in their surrounding. Increased mortality effect of reference sample was also observed for the cell grown in non-direct contact with the samples. Cell cycle analysis showed the highest cell mortality in the surrounding of reference, 5% and 10% OCTAPOSS samples. After 72 hours of culture, the mildest toxic effect was observed for the 10% PHIPOSS sample. Proliferative activity potential, defined by the number of cells in S phase, was systematically decreased along with the culture time increase, in both-cells grown on the sample and in its surrounding. Apoptosis/necrosis evaluation with Annexine V Experiment with Annexine V conjugated to fluorescein (FITC) and propidium iodine (PI) was designed to determine the pathway of cell death, during culture with the test samples. The test applied allows to distinguish between sudden, uncontrolled necrosis and strictly controlled, runned in successive stages apoptosis. Cells showing only PI fluorescence were considered necrotic, cells emitting only FITC fluorescence as early apoptotic, cells simultaneously showing PI and FITC were considered to be late apoptotic, and cells that did not show any fluorescence were considered as viable cells (Fig. 11).

Fig. 11. Number of cells in a manner of different types of cell death: necrosis-(PI fluorescence emission), early apoptosis (only FITC emission) and late apoptosis (PI and FITC emission).

Comparison of cells grown on the material surface vs cells grown in their surrounding. Our study confirmed the high mortality ratio of U-2 OS osteoblasts grown in both: direct contact with samples of the tested materials and in their environment. Comparing to control plate, cells growing in direct contact with the tested samples, as well as in their environment, showed increased mortality (Fig. 12).

Fig. 12. Comparison of number of cells in S phase (viable) and apoptosis (dead cells). Cells grown in direct contact with the sponges tested vs cell grown in the surrounding of the material in time.

The analysis carried out indicated, that the changes observed had an abrupt character. Cells grown directly on the test samples after 24 hours were presenting larger percentage of late apoptotis within the population, while at the same time cells grown on the surface of the tested materials were subject to necrosis more frequently (Fig. 13).

Fig. 13. Fluorescence intensity of apoptotic (Annexin V) vs necrotic (propidium ioide) cells grown in contact with materials tested for 24, 48 and 72 hours.

The viability changes profile of the examined cells has changed during the test and at 72 hours osteoblasts grown on the test samples, as well as in their surrounding, were in the late apoptosis. The mortality of U-2 OS cells grown directly on the reference material was very high and had an abrupt character.

The cellular effects observed can be related to properties of tested polymers surface. Polyurethane foams have strong hydrophobic properties and poorly absorb water. According to our previous work [29], it is related to high material density, which strengthens its mechanical properties. What is more, hydrophobic properties of tested samples were further enhanced by POSS molecules. High hydrophilicity of biomaterials promotes cell adhesion to sample surface and allows their proliferation [42, 43]. Hence, hydrophobic character of polyurethane foams tested may be the cause of limited cell viability grown in direct contact with the tested materials. Direct correlation between adhesion and proliferative activity, reduction of adhesion to the medium, negatively affects the growth potential of cells. Consequently, cells growing in surrounding of the tested samples showed lower mortality ratio, as they could adhere to the surface of the culture vessel. Moreover, the materials tested contain POSS molecules in their structure and it was already indicated that they might be responsible for formation of Reactive Oxygen Species (ROS) and indirectly cause destruction of cell and mitochondrial DNA, resulting in cell death [44]. They can also directly enter cell membranes, bind to DNA and lead to its degradation [45, 46]. 4. Conclusions Incorporation of functionalized POSS moieties in flexible polyurethane foams as pendant groups (PHIPOSS) and chemical crosslinks (OCTAPOSS) leads to (micro)structural changes composed to unmodified polyurethane foams. It has been observed that with an increase of the filler the open cell structure formation is facilitated as crystals formed on the surface of modified materials may contribute to cells’ opening. The distribution maps show that silicon is dispersed in the entire sample volume. In the case of PUF-OCTAPOSS systems, on the surface of the material, there are few, fuzzy areas of OCTA-POSS agglomerates that are not visible on WAXD patterns. In contrast, in PUFPHIPOSS numerous crystals are formed in the shape of characteristic "crosses", which are evenly distributed over the entire surface of the material. The micro tomography results showed that the introduction of these POSS molecules into the polyurethane foam leads to an increases in the number of pores in the volume unit. The incorporation of OCTAPOSS molecules into the polyurethane structure as nodes of the polymer network, regardless of the content introduced, did not result in the appearance of additional peaks on the obtained WAXD diffractograms. Different results were recorded when PHIPOSS molecules are built into the polyurethane structure. When the PHIPOSS content in the PUF is 10 or 15 wt. %, reflexes appear at the deflection angle of 2θ = 8.1°, corresponding to the most intense diffraction peak of PHIPOSS, which confirms the presence of its crystallites. The bioactivity of PUF-POSS composites was confirmed on the basis of their behavior in SBF, leading to the formation of hydroxyapatite on the foams surfaces, which provided the basis for conducting research to assess the biocompatibility of PUF-POSS materials. The investigations showed that PUF-POSS materials negatively affected the cell viability of the U-2 OS osteoblasts. These effects were primarily visible for the reference sample. Foams modification with OCTAPOSS and PHI-POSS did not completely eliminate the adverse effect, however, compared to the reference samples, a reduction in the cytotoxic effect was observed. In the analysis of the cell cycle of osteoblasts U-2 OS, a noticeably smaller percentage of dead cells was observed in cultures with the majority of modified samples compared to the reference foam. This effect is visible in both: cells grown directly on samples of materials, as well as in their environment. When comparing both types of modifications, PHI-POSS additives presents better bioadaptation properties. Declaration of Competing

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- describes the novel flexible polyurethane foam hybrids (PUFs) chemically modified by functionalized polyhedral oligomeric silsesquioxanes (POSS) - shows that incorporation of different POSS nanoparticles into PUF changes their structure and properties - evaluates the effect of POSS particles on the cytotoxicity of flexible polyurethane foams

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: